The goal of this proposal is to further understand the role of prdm1a and genes it modulates during craniofacial development. Prdm1a is a transcription factor that I hypothesize to be a key regulator of neural crest specific genes during posterior craniofacial development. To further understand the role of prdm1a in posterior craniofacial development it is imperative to identify direct downstream targets to begin to build a regulatory network of genes regulated by Prdm1a. I hypothesize that prdm1a modulates and finely tunes the expression of multiple neural crest specific genes in concert to orchestrate proper development of the posterior craniofacial skeleton. To test this, I will analyze potential downstream targets of Prdm1a identified in our microarray using Chromatin Immunoprecipitation (ChIP) to probe for Prdm1a direct binding of targets of interest. A knockdown approach, using morpholinos, will be used to perturb gene function to determine if target knockdown phenocopies prdm1a mutant zebrafish. Rescue experiments will be performed using mRNA injection into prdm1a mutant zebrafish to determine if target mRNA can rescue the mutant phenotype. I will utilize ChIP-seq to identify novel targets of Prdm1a followed by in situ hybridization (ISH) of 5-10 targets of interest to confirm localization to the craniofacial region. Furthermore, gain and loss of function experiments will also be performed to investigate function of potential targets. Lastly, to further build the GRN of the posterior face, I am screening the F2 generation from an ENU mutagenesis screen to identify novel posterior craniofacial mutants to continue to build the posterior craniofacial GRN. The overarching goal of this proposal is further elucidate the role of prdm1a during craniofacial development by building a gene regulatory network for prdm1a through identification of direct downstream targets, coupled with identification of novel regulators of posterior craniofacial development. The techniques and experiments described will aid me in testing my hypothesis. Furthermore these studies can be applied to other genes involved in craniofacial development to build a more comprehensive gene regulatory network which is necessary for understanding how and why these syndromes occur, and more importantly how to design potential therapies and treatments. PUBLIC HEALTH RELEVANCE: Neural crest cell defects are implicated in many syndromes resulting in cleft lip and palate in approximately 6800 live births in the United States alone (March of Dimes, 2010). Understanding the genes involved in neural crest differentiation, specification, and migration during craniofacial development is a critical step in developing potential therapies and treatment for craniofacial disorders.